AU626797B2 - Immunogenic composites capable of selectively inducing antibody production, pharmaceutical compositions employing the same and method of selectively inducing antibody production - Google Patents

Description

rk

-V

P1/00/01 1A 1 j~fc~cL Form PATENTS ACT 1952-1973 COMPLETE SPECIFICATION

(ORIGINAL)

FOR OFFICE USE 626797 Class: Int. C Application Number: Lodged: 9*

E

Complete Specification-Lodged: Accepted: Published: Priority: Related Art: S TO BE COMPLETED BY APPLICANT Name of Applicant: *,o Address of Applicant: Actual Inventor:s Address for Service: ALBANY MEDICAL COLLEGE, a corporation of the State of New York, of 47 New Scotland Avenue, Albany, New York, 12208, United States of America.

Raphael J. MANNINO and Gail GOODMAN-SNITKOF Care of JAMES M. LAWRIE CO., Patent Attorneys, of 72 Willsmere Road, Kew, Victoria, 3101, Australia.

Complete Specification for the invention entitled: IMMUNOGENIC COMPOSITES CAPABLE OF SELECTIVELY INDUCING ANTIBODY PRODUCTION, PHARMACEUTICAL COMPOSITIONS EMPLOYING THE SAME AND METHOD OF SELECTIVELY INDUCING ANTIBODY PRODUCTION The following statement is a full description of this invention, including the best method of performing it known to me:-* "Note: The description is to be typed in doublo spacing, pica type face, in an area not exceeding 250 mm In depth and 160 mm In width, on tough white paper of good quality and it is to be Inserted inside this form.

11710/76-L I iiI, l I (htr n ,,tnr rinte. Canbrra LIMMUNOGENIC COMPOSITES CAPABLE OF SELECTIVELY INDUCING ANTIBODY PRODUCTION, PHARMACEUTICAL COMPOSITIONS EMPLOYING THE SAME AND METHOD OF SELECTIVELY INDUCING ANTIBODY PRODUCTION FIELD OF THE INVENTION The present invention is directed to immunogenic composites capable of inducing selective antibody production. In particular, the present invention is directed to immunogenic composites capable of selectively inducing antibody production to both synthetic and naturally.occurring peptides without the need to link the peptides to a heterologous protein carrier BSA or KLH) and without the need for the peptides to be injected in conjunction with additional adjuvants. The present invention is also directed to a pharmaceutical composition employing the immunogenic composites and to a method for selectively inducing antibody production employing the pharmaceutical composition.

0.

BACKGROUND OF THE INVENTION The development of effective vaccines is of significant importance in the control and prnphblaxis of diseases. The use of synthetic peptides as immunogens is being intensively investigated ac one promising approach to the preparation of safe and effective subunit vaccines (Lerner, Green. N..

Alexander, Liu, Sutcliffe, and Shinnick, T.M. Proc. Natl. Acad. Sci. USA 78:3403-3407 (1981); Bhatnagar, Papas, Blum, Milich.

Nitecki, D. Karels, and Vyas, G.N Proc.

It'.

Natl. Acad. Sci. USA 79:4400-4404 (1982); Neurath, Kent, and Strick, N. J. Gen. Virol..

65:1009-1014 (1984); Dreesrnan, Sparrow, J.T., Frenchick, and Kennedy, R.C. Adv. Exp,_Med. Biol..

185:129-137 (1985); Thanavala, Brown, S.E., Howard, Roitt, and Steward, M.W. J. -Ep Med. 164:227-236 (1986); Muller, Shapira, and Arnon, R. Proc. Natl. Acad. Sci. USA 79:569-573 (1982); Kennedy, Henkel, Pauletti, Allan, J.S., Lee, Essex, M. and Dreesman, G.R. Science 231:1556-1559 (1986); Bittle, Houghten. R.A., Alexander, Shinnick, Sutcliffe, Lerner, Rowlands, D.J. and Brown, F. Nature 298:30-33 (1982) and DiMarchi, Brooke, Gale. C., Cracknell, V. Doel, T. and Mowat, N. Science 232:639-641 (1986)). However, the development of synthetic peptide vaccines has been hampered by the need for both carriers and adjuvants which frequiently have -undesirable side effects (Lerner, C-re-n. N., Alexander, Liu, Sutcliffe, J.G, and Shinnick, T.M. Proc. Natl. Acad. Sci. USA 78:34nl3-3407 19 81) Bha tnaga r, P. K. P apa s, E. B lum, H. E. ,M iIi ch, D. Nitecki, D. Karels, M. J. and Vyas, G. N Proc-.

Nati. Acad. Sci. USA 79:4400-4404 (1982): N-'trath, Kent, and Strick, H. J. Gen. Virol.

65:1009-1014 (1984); and Muller, Shapira, and Arnon, R. Proc. Natl. Acad. Sci. USA 79:5f;9-573 (1982)). In addition, immunization with peptide-protein carrier complexes usually r,-'sis int 4 the priming of the T cells to the foreign rarrier.

Hence, a natural infection does not produce a sprnndary 2- 1.1-111-1- -V c*;

S

C.

0* S

S

*S 5 S C

S

C

S@ S j immune response since the animal's T cells are not primed to the natural carrier of the neutralizing epitope.

Accordingly, there still exists a need for effective immunogens which do not require that the peptides be linked to heterologous protein carriers and do not require injection in conjunction with additional adjuvants.

SUMMARY OF THE INVENTION One object of the present invention is to provide an immunogenic composite and a pharmaceutical composition that simulates the effectiveness of natural infection or vaccination with the currently available procedures (including inactivated or attenuated agents, or macromolecules, or subunits thereof, from the outer surface of infectious agents), without any of the undesirable side effects of the currently available preparations.

A second object of the present invention is to provide an immunogenic composite and a pharmacretical composition that avoids the problem of priming T cells specific to a heterologous carrier which w-iild not result in a response during subsequent natural exposure.

A third object of the present invention is to provide a method for priming homologous helper T cells which will respond quickly during the natural inf-ction resulting in the secondary immune response.

3 ,1 These and other objects have been achieved by providing an immunogenic composite capable of selectively inducing or enhancing antibody production.

In a first embodiment, the present invention provides an immunogenic composite capable of selectively inducing or enhancing antibody production to a non-immunogenic peptide comprising a peptide-lipid complex which comprises a non-immunogenic amphipathic peptide covalently bound to a lipid, under the proviso that the amphipathic peptide is not albumin. In a preferred embodiment, the peptide-lipid complex is associated with a mixture of one or more lipids and one or more sterols.

In a second embodiment, the present invention provides an immunogenic composite capable of selectively inducing or enhancing antibody production to one or more non-immunogenic peptides comprising a peptide-lipid complex which comprises a hybrid peptide covalently bound to a lipid and said hybrid peptide comprises a non-immunogenic amphipathic peptide covalently bound to one peptide selected from the group consisting of: a non-immunogenic hydrophilic peptide; a non-immunogenic neutral peptide; a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two or more of the above-defined peptides and under the proviso that the amphipathic peptide is not albumin. In a preferred embodiment the peptide-lipid complex is associated with a mixture of one or more lipids and one or more sterols.

In a third embodiment, the present invention provides an immunogenic composite capable of selectively inducing or enhancing antibody production i I4**

-~III~U~L~

V to one or more non-immunogenic peptides comprising a first peptide-lipid complex and one or more additional peptide-lipid complexes associated with a mixture of one or more lipids and one or more sterols, wherein said first peptide-lipid complex comprises one member selected from the group consisting of: a non-immunogenic amphipathic peptide covalently bound to a lipid; and a hybrid peptide covalently bound to a lipid, wherein said hybrid peptide comprises a non-immunogenic amphipathic peptide covalently bound to a peptide selected from the group consisting of: a non-immunogenic hydrophilic peptide; a non-immunogenic neutral peptide; a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two or more of the above-defined peptides. and and said one or more additional peptide-lipid complexes comprises one or more members selected from the group consisting of: a non-immunogenic hydrnphilic peptide covalently bound to a lipid; a non-immunogenic neutral peptide covalently bound to a lipid; a non-immunogenic amphipathic peptide covalently bound to a lipid, and a hybrid ppptide covalently bound to a lipid, wherein said hybrid peptide comprises a non-immunogenic amphipthic, hydrophilic or neutral peptide covalently brnind to a peptide selected from the group consisting of: a non-immunogenic hydrophilic peptide; a non-immunogenic neutral peptide; a non-immnngenic amphipathic peptide; and a hybrid peptide 5 jl comprising any combination of two or more of the above-defined peptides and under the proviso that the amphipathic peptide is not albumin.

The present invention also provides a pharmaceutical composition for selectively inducing or enhancing the production of antibodies which comprises a S 5 mixture of one or more of the above-described immunogenic composites and a I pharmaceutically acceptable carrier, diluent or excipient, under the proviso that the amphipathic peptide is not albumin.

In a further embodiment, the present invention provides a method for selectively inducing or enhancing antibody production comprising administering to a host, in which an immune response is the normal means of defense to infection by foreign materials, a pharmaceutically effective amount of the abovedescribed pharmaceutical composition.

*0 Be p ee -6- 9 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the primary amino acid sequence of various peptides useful in the immunogenic composites of the present invention.

Figures 2, 3, 4 and 5 are profiles derived from Hopp and Woods' analysis of peptides useful in the immunogenic composites of the present invention: Figure 2 shows profiles for amphipathic peptides.

HIV(469-511) and HIV(487-511) and for hydrophilic peptides HIV(469-485) and HIV(500-511); Figure 3 shows a profile for the amphipathic peptide HIV(578-608); ee Figure 4 shows a profile for the hydrophilic peptide HIV(647-659); and Figure 5 shows a profile for the neutral peptide (NANP) The asterisks represent the degree of hydrophilicity or hydrophobicity, hydrophilic being above the single letter amino acid code and hydrophobic being below the single letter aminn acid code. The greater the number of asterisks, the greater the hydrophilicity/hydrophobicity.

7 -7i DETAILED DESCRIPTION OF THE INVENTIO0N The alphabetical nomenclature used herein for describing peptide sequences is the standard amino acid alphabet set forth below.

Three-letter One-letter Amino Acid abbreviation abbreviation IAlanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D **Asparagine or Asx B aspartic acid Cysteine Cys C Glutamine Gln9 Glutamic Acid Glu E Glutamine or Glx Z glutarnic acid Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methion-ine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y *Valine Val V For purposes of the present invenion. the following terms have the definitions set forth bp1ow.

V

toww*- Peptide A series of amino acids polymerized head to tail by a peptide bond between the carboxylic acid group of one and the amino acid group of the next. The peptide can be naturally occurring, recombinantly produced, synthetically produced or derived by chemical or enzymatic cleavage of a large polypeptide or protein so obtained, or by chemical or enzymatic buildup of a peptide from smaller units and the peptide can have moieties other than only amino acids, such as sugars, or can have various chemical modifications such as, for example, phosphorylation, methylation, sulfation or acetylation, these mofidications occurring as a result of their production, for example in microorganisms, or by affirmative alteration using organic chemical or enzymatic synthetic techniques.

Non-immunoqenic peptide A peptide, as defined above, but that when administered to an animal without being conjugated to or mixed with additional substances does not stimulate an immune response in that animal.

(Some peptides can be immunogenic in one animal but non-immunogenic in an animal of a different allntype.) Additionally, as used herein, the term "non-immunngenic peptide" can include peptides that are immunogpnir, but whose immunogenicity is enhanced by formation nf an "immunogenic composite" according to the present invention.

Amphipathic peptide A peptide, as defined above, but that contains distinct hydrophobic and hydrnphilic regions. Examples of amphipathic peptides are. (1) peptides that contain contiguous stretches of mainly hydrophobic amino acids followed by rcntiquous -9 stretches of mainly hydrophilic amino acids, or (2) peptides that can assume an alpha-helical structure in solution such that one side of the alpha helix comprises mainly hydrophobic amino acids and the other side of the alpha helix comprises mainly hydrophilic amino acids. The amphipathicity of the second type of peptides is said to have alpha helical periodicity.

The hydrophobic/hydrophilic nature of the regions is defined by computer analysis using programs that take into account the hydrophobic/hydrophilic properties of amino acid side chain residues.

Examples of such programs which can be applied to

*S

determine the existance of hydrophobic/hydrophilic S stretches are those developed by Hopp and Woods (Hopp T.P. and Woods, K.R. Proc. Natl. Acad. Sci. USA 78:3824-3827 (1981) and Hopp T.P. and Woods, K.R. Mol.

Immunol. 20:483-489 (1983)) or Kyte and Doolittle (Kyte, J. and Doolittle, J. Mol, Biol.

*0 157:105-131 (1982)) although other analogous programs can also be used.

Computer programs have also been developed which

S

can determine the amphipathic nature of a peptide should the peptide form an alpha helical structre in solution. In this instance, a distinct hydrophilic region refers to one side of the alpha helical ppptide.

while a distinct hydrophobic region refers to the other side of the peptide. An example of such a program is provided by Delisi and Berzofsky, Proc.

Natl. Acad. Sci. 82:7048 (1985).

Also, for the purposes of this invpntion, a hydrophobic, hydrophilic or neutral region is d-fined ii 2 ~c~nt~ 0 0*0 S @0 6 0S 0* 0000 0

S

6* S 0 *6 @0 0

S@

*4 0 0 *09 00 by computer analysis, either of the primary sequence or with respect.to alpha helical periodicity as described above.

One physical test of amphipathicity is the ability to perturb the integrity of a phospholipid membrane.

This property can be tested by measuring the ability of the peptide to promote the release of carboxyfluorescein from the internal aqueous space of preformed liposomes as described below. However, not all amphipathic peptides will necessarily perturb the integrity of a phospholipid membrane, and, accordingly, for the purposes of this invention perturbation of a phospholipid membrane is not an absolute requirement of the definition of'an amphipathic peptide.

Hydrophobic peptide A peptide, as defined above, but that is hydrophobic in a Hopp and Woods-type or Kyte and Doolittle-type analysis for contiguous stretches or a Delisi and Berzofsky-type analysis for alpha helical periodicity as described above.

Hydrophilic peptide A peptide, as defined above.

but that is hydrophilic in a Hopp and Woods-type or Kyte and Doolittle-type analysis for cnnt iuous stretches or a Delisi and Berzofsky-type analysis for alpha helical periodicity as described above.

Neutral peptide A peptide, as defined ahn but that is neutral in a Hopp and Woods-type or Kyte and Doolittle-type analysis for contiguous stretrchp- or a Delisi and Berzofsky-type analysis for alpha helical periodicity as described above.

-11 SAccording to the present invention, at least three general embodiments of the immunogenic composite are envisioned.

A first embodiment provides an immunogenic composite capable of selectively inducing or enhancing antibody production to a non-immunogenic amphipathic peptide. The second and third embodiments provide immunogenic composites capable of selectively inducing or enhancing antibody production to a non-immunogenic hydrophilic and/or a non-immunogenic neutral peptide as well as to a non-immunogenic amphipathic peptide.

The composite according to the first embodiment comprises a peptide-lipid complex comprising a non-immunogenic amphipathic peptide covalently bound to a lipid, under the proviso that the amphipathic peptide is not albumin. In a preferred embodiment, the peptide-lipid complex in associated with a mixture of one or more lipids and one or more sterols.

The peptide-lipid complex is prepared by covalently bonding an amphipathic peptide to a suitable lipid according to numerous known means such as, for example, by cross-linking to glysphingolipids (Heath, et al. B.B.A.

i 640:66-81 (1981)); or via N-(p-aminophenyl)sterylamide (Snyder, S.L. and Vannier, W.E. B.B.A. 772:288-294 (1984)); or via the N-hydroxysuccinimide ester or palmitic acid (Huang. A. et al. T.B.C. 255:8015-8018 (1980)).

Covalently binding the peptide to the lipid by cross-linking is preferred and can be accomplished by methods well known in the art, for example, by a method using N-succinimidyl-4-(p-maleimidophenyl)butyrate S-12- (Martin, and Papahadjopoulos, D. J. Bia. Chem.

257:286-288 (1982)); N-hydroxysuccinimidyl 3-(2--pyridyldithio) propionate (Barbet, J. et al. J. Supra!.

Struct. and Cell Biochem. 16:243-258 (1981)); rn-maleimidobenzoyl-N-hydroxysuccinimide ester (Hashimoto, Y.

et al. J. Immuno. Methods 62:155-162 (1983)); citraconylation (Jansons, V.E. and Mallett, P.r. Anal..

Biochem. 111:54-59 (1981)), As the peptide, any amphipathic peptide that meets the above definition can be used. of course, if the immunogenic complex is to be used as a vaccin., the peptide should not be toxic or otherwise detrimental to the health of the animal.

*:se Further, as mentioned above, amphipathicity can be tested by measuring the ability of the pepf-iie to promote the release of carboxyfluorescein from the internal aqueous space of preformed liposomes (Szoka and Papahadjopoulos, Proc. Nat. Acad. Sci. USA 0 75:4194-4198 (1978) and Weinstein, J.N. et al. Science 195:489-491 (1977)), although this is not a ne-essary test for purposes of the present invention.

Specifically, carboxyfluorescein (rF) is encapsulated in large unilammelar pospholipii v.eicles by known methods (Szoka and Papahadjopoul-s. Proc.

Natl. Acad. Sci. USA 75:4194-4198 (1978)) a t self quenching concentrations (-200mM, Weinstein. JT N. et al., Science 195.489-491 (1977)). CF is puirified by known methods as described by Ralston, et al (Biochim.

Biophys. Acta. 649:133-137 (1981)). The ahi Ii ty of peptides to perturb the integrity of the 1ipnsomal bilayer can be determined by measuring emission At 520 -13- II;I II r r nm 30 minutes after the addition of peptide to a suspension of liposomes. In general, an increase in fluorescence greater than 5% of the total possible increase is indicative of amphipathicity. Total possible fluorescence increase can be determined by measuring emission at 520 nm before and after lysis of vesicles with 0.1% Triton X-100.

Examples of amphipathic peptides include the following peptides derived from the human immuno deficiency virus, HIV, envelope protein: HIV(487-511), HIV(469-511) and HIV(578-608) (Starcich et al. (Cell 45:637-648 (1986)). Further, antibodies to the following sequences recognize the HIV surface I glycoprotein or the sequences are included in a region i to which neutralizing antibodies are produced: HIV(735-752)(D-R-P-E-G-I-E-E-E-G-G-E-R-D-R-S-NH2' Kennedy et al., Science 231:1556-1559 (March 1986)).

SHIV(340-368)(N-N-T-L-K-Q-I-D-S-K-L-R-E-Q-F-G-N-N-L-Q- S-S-G-C-NH2) alone or HIV(299-329)(H-R-P-N-N-N T-R-K- 22 I-R-I-E-R-E-P-E-R-A-E-K-I-E-N-M-R-Q-C-NH wit h an amphipathic peptide of HIV 487-511) to enhance antibody production. Both HIV(340-36P) and HIV(299-329) are from a region of the viru~ which produces neutralizing antibodies. (Putney P- al., Science 234:1392-1395 (Dec. 1986)).

The primary sequences of HIV(487-511). HIV- (469-511) and HIV(578-608) are set forth in Figure 1.

Hopp and Woods profiles of HIV(487-511) and HIV(469-511) are shown in Figure 2 and a Hopp and Woods profile of HIV(578-608) is shown in Figure 3.

-14-

V

I 1 As the lipid that is covalently bound to the amphipathic peptide, any lipid can be used, providing that means can be found to covalently couple the peptide. to the lipid. One skilled in the art can readily determine if such means are present.

SExamples of suitable lipids include phospholipids such as phosphatidylethanolamine sterols such as cholesterol; spingolipids such as sphingomyelin; glycolipids such as myeline; and other diacyl containing lipid structures, diacylamines.

j Phospholipids are preferred, and *phosphatidylethanolamine is especially preferred.

i The peptide-lipid complex can be purified for use i by removing unreacted components and detergent by dialysis. The material remaining in the dialysis bag after dialysis is the immunogen. No further purification needs to be performed.

In addition to preparing a peptide-lipid complex by covalently bonding the peptide to the lipid, naturally occuring complexes can also he used.

Examples of such naturally orriring peptide-phospholipid complexes are complexes r-mprised Sof proteins linked to phosphatidylinositol.

S The above-described peptide-lipid complex an be used alone as the immunogenic composite acrc-rni ng to the present invention. However, in a preferred embodiment, the peptide-lipid complex is assn-iated with a mixture of one or more lipids and one or more sterols.

A ,The composite comprised of a peptide-lipid rnmplex associated with a mixture of one or more lipids and one j or more sterols can be produced by solubilizing all components, i.e. the peptide-lipid complex, the additional lipids, and the sterols, in a nnntoxic detergent with a high critical micelle concentration.

This solution is then dialyzed to remove the detergent.

I The result is a particulate suspension that is either vesicular in nature or what appears to be, when viewed by light microscopy, an amorphous particulate structure.

While not wanting to be bound by the following S explanation, the nature of the peptide in the composite appears to be the factor determining the structure that results. That is, those peptides that are hydrnphilic S or neutral appear to more often form vesicular I. structures whereas those that are amphipathic appear to S more often form a',iorphous, particulate aggregates.

The peptides are most probably associated with each other and with the additional lipids and sterols via hydrophobic interactions resulting from the acyl chains of the lipid to which the peptide is cnni;ngated and the hydrophobic regions of the other lipirls and sterols. However, as with membrane structures. ionic interactions and Van der Waals forces may also be involved.

Association can be tested by centrifugation -f the 1 dialyzed solution in an ultracentrifuge at higi speed (100,000 300,000 xg). If the peptide-lipid complexes have been associated with the additional lipids and sterols to form an immunogenic composite accnrding to the present invention, the peptide-lipid complox p will pellet along with the additional lipids and sternls.

-16 I I i

S

S. S

S

S.

S.

S

S.

S

5

S..

The fate of peptide-lipid complexes can be monitored by, for example, radioactive label, fluorescent label, spectroscopy, thin layer chromatography or high pressure liquid chromatography.

More specifically, there are at least two ways to produce the composite comprised of a peptide-lipid complex associated with a mixture of one or more lipids and one or more sterols.

According to one method, derivatized lipid (for forming the peptide-lipid complex) is dissolved in detergent along with a mixture of one or more nonderivatized lipids and one or more sterols. A premade activated peptide is then added to the solution and allowed to react with the derivatized lipid to form a peptide-lipid complex. After completion of the reaction, the solution is dialyzed against an appropriate aqueous buffer to remove the detergent thereby forming a particulate suspension comprising the immunogenic composite.

The derivatized lipid for use in the above-described method is prepared accordina to conventional means such as those described for producing the peptide-lipid complex by itself For example, if a cross-linking agent is used to f-rm the peptide-lipid complex, the lipid is derivati7pd by joining a cross-linking reagent to the lipid. If necessary, the derivatized lipid is then extracted from the reaction mixture, also by conventional means.

The peptide is activated in accordance with the means used for joining the peptide to the lipid For example, if a cross-linking agent is used to jnin the -17 6b.- J_ Speptide to the lipid, the peptide is reduced with dithiothreitol by conventional means. The thus activated peptide is purified by conventional means such as affinity chromatography, or HPLC (high pressure liquid chromatography).

The detergent in which the thus-prepared derivatized lipid is dissolved along with one or more nonderivatized lipids and one or more sterols can be any nontoxic detergent with a high critical micelle concentration. By "high" critical micelle concentration, it is meant that the detergent can be o removed by dialysis. Other detergents with "low" S" critical micelle concentrations, such as Triton X-100 e or C 1 2

E

8 (N-dodecyloctaethylene monether), are also suitable but removal must be accomplished by means other than dialysis, such as, for example, by adsorbtion onto SM-2 beads. Suitable detergents having a high critical micelle concentration can readily be determined by one skilled in the art and examples include octyl-B-D-glucoside and octyl-S-D-thingl iiopyranoside.

Octvl-B-D-glucoside is preferred.

£he lipids and sterols are prepared for solubilization in detergent by first dissolvinq in an organic solvent that solubilizes but does no- nxidize or in any way destroy the integrity nF the phospholipid. Suitable organic solvents include diethyl ether, chloroform, benzene, and acetone.

The derivatized lipid, non-derivatized lipids. and ste-ols are used in a molar ratio of about 2:3:5.

although a broad range of ratios can be used, and -18 dissolved in the organic solvent at a concentration of about 10 mg (lipid and sterol)/ml (solvent), although a range of concentration ratios can be used.

The sample is then dried under a non-oxidizing atmosphere such as a nitrogen stream or argon.

The dried sample is resuspended in a suitable aqueous buffer such as an aqueous phosphate buffer or an aqueous citrate buffer, pH 4.5 to 6.5, at a concentration of about 4 mg(lipid and sterol)/1 ml (buffer) and the sample is sonicated at room temperature to form a particulate suspension of vesicle.

oThe detergent is then added to this sample and the sample is sonicated again at room temperature to dissolve all components.

SThe amount of detergent added is enough to solubilize the components in the mixture. A suitable .o amount is about 10:1 by weight based on the weight of total lipids and sterols. One skilled in the art can readily determine other suitable amounts of detergent.

To form the peptide-lipid complex, the activated peptide, prepared as described above, is added to the detergent mixture in an amount necessary to optimize coupling of the peptide to the derivatized lipid, for example in an amount of about 1:2 moles peptide per mole derivatized lipid. The pH and other reaction conditions are adjusted in accordance with the particular reaction to be carried out, and the peptide and derivatized lipid are allowed to react appropriately. The reaction conditions are readily -19 P_ determined by the skilled artisan. A suitable reaction time is generally overnight.

The dialysis is carried out according to conventional means for a period of time and with enough changes of buffer to remove essentially all of the detergent.

As the dialysis buffer, any aqueous buffer can be used that does not oxidize lipid or destroy the integrity of the immunogenic composite.

The result is a particulate suspension of the immunogenic composite comprising a peptide-lipid complex associated with a mixture of one or more lipids and one or more sterols.

According to a second method for preparing the S immunogenic composite comprising a peptide-lipid complex associated with a mixture of one or more lipids and one or more sterols, a premade activated peptide, prepared as described for the first method, is added to a suspension containing vesicles comprised of derivatized lipid, also prepared as described for the first method, and a mixture of one or more lipids and one or more sterols, and the peptide is allowed to react with the ieed lipid in the vesicles to form a peptide-lipid complex. An immunogenic composite with a peptide-lipid complex already in it can also be used as vesicles. After completion of the r ction, all of the components are solubilized in detergent, and the solution is dialyzed, as in the first method, against an appropriate aqueous buffer to remove detergent thereby forming a particulate suspension comprising the immunogenic composite.

V

-ii--i I 0 In the second method the solution containing unilamellar vesicles is prepared in the same manner as the particulate suspension of vesicles is prepared in the first method. Peptide is reacted as above prior to the addition of detergent. Detergent is added at the end of the dvernight coupling reaction. All other steps, times, concentrations, temperatures, pH's etc., are identical to those described in the first method.

More specifically, to form the peptide-lipid complex, the activated peptide is added to the mixture in an amount necessary to optimize coupling of the peptide to the derivatized lipid, for example in an S, amount of about 1:2 moles peptide per mole derivatized S* lipid. The pH and other reaction conditions are adjusted in accordance with the particular reaction to I be carried out. The reaction conditions are readily determined by the skilled artisan. A suitable reaction time is generally overnight.

SThe detergent used to solubilize the components in the reaction mixture is the same as described abn e for the first method.

The sample is prepared for solubilization by adding detergent to the reaction mixture in an amount sufficient to solubilize the componetns of the mixture.

A ratio of about 10 mg detergent to 1 mg tnt-al lipid and sterol is generally suitable. The sampJ] is then sonicated to dissolve all components.

Once solubilized in detergent, dialysis is rcrried out as described above for the first method.

As the lipid and the peptide to be used in this embodiment of the invention, any lipid and any -21 rrrr, amphipathic peptide as described above for preparing the immunogenic composite comprising only an amphipathic pepfi-de covaln tf b'ound to a -lipid can be used.

ospholipids are preferred and phosphatidy]ethanolamine is especially preferred.

The lipid component of the lipid/sterol mixture can be any lipid; providing it is not immunogenic, i.e.

an animal does not demonstrate an immune response to the lipid either alone or in the composite. Nor should the lipid interfere with the ability of an animal to S* m mount an immune response to_the-peptide.

One skilled in the art can readily determine *0 0 whether a lipid is immunogenic by assaying for antibodies reactive against lipid in standard assays.

I Further, the lipid component of the lipid/sterol mixture can be comprised of all one type of lipid or a mixture of two or more lipids, and the lipid may be the same as that present in the protein-lipid complex or it may be different.

Examples of lipids useful as the lipid component of the lipid/sterol mixture include phosphatidylserine phosphatidylcholine spingomye]in (SP).

phosphatidylethanolamine phosphatidylinnsito.

phosphatidylglycerol phosphatidir acid and cardiolipin.

Phospholipids are preferred.

As the sterol, any sterol can be used. pr~-idina that it does not solubilize lipid assemblies and is not immunogenic, it does not induce an immune response against itself and does not interfere with the -22 Sit 'i ability of an animal to respond to the peptide, for example possess anti-inflammatory properties.

Mixtures of more than one sterol can be used, but use of only one sterol is preferred.

As used in the context of the lipid/sterol mixture, sterol means a class of abundant steroids containing an alcoholic hydroxyl group at the C 3 carbon and a branched aliphatic chain of 8 or more carbon atoms at the C 17 carbon. They occur either as free alcohols or as long chain fatty acid esters of the Shydroxyl group at the C 3 carbon.

.3 Examples of sterols that can be used in the mixture include cholesterol and lanosterol.

An especially preferred sterol is cholesterol.

The immunogenic composites, in second and third embodiments, are capable of selectively inducing or enhancing antibody production to a non-immunogenic hydrophilic and/or a non-immunogenic neutral peptide.

as well as to a non-immunogenic amphipathic peptide.

One of these two immunogenic composites romptises a peptide-lipid complex which comprises a hybrid peptide covalently bound to, a lipid and said hybrid peptide comprises a non-immunogenic amphipathir ppptide covalently bound to one peptide selected from the group consisting of: a non-immunogenic hydrophilic peptide; a non-immunogenic neutral peptide: a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two or more of the above-defined peptides and In a preferred embodiment, the peptide-lipid complex is -23

!-E

t, i associated with a mixture of one or more lipids and one or more sterols.

This immunogenic composite, either the peptide-lipid complex alone or associated with a mixture of one or more lipids and one or more sterols, can be prepared in the same manner as described above for the immunogenic composite comprising a non-immunogenic amphipathic peptide covalently bound to a lipid, except that the peptide in the peptide-lipid complex comprises a hybrid peptide comprising a non-immunogenic amphipathic peptide covalently bound to one of: a non-immunogenic hydrophilic peptide; a non-immunogenic neutral peptide; a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two or more of the above defined peptides (b) and under the proviso that the amphipathic peptide is not albumin.

The peptides forming either of the hybrid peptides are covalently bonded together, preferably end-to-end.

Further, since amino to amino and carboxyl to carboxyl bonds are usually unstable in an aqueous environment, end-to-end bonding is preferably by amino ends to carboxyl ends.

i :As the hydrophilic or neutral peptide, any hydrophilic or neutral peptide that meets the above definitions can be used. Also, if the immunogenic complex is to be used as a vaccine, the peptide should not be toxic or otherwise detrimental to the health of the animal.

Examples of hydrophilic peptides include HIV(469-485), HIV(500-511) and HIV(647-659).

*2 ~w~sFI 5-24- -1 *e~ 2* C The primary sequences of these peptides are shown in Figure 1.

Hopp and Woods profiles of HIV(469-485) and HIV(500-511) are shown in Figure 2 and a Hopp and Woods profile of HIV(647-659) is shown in Figure 4.

!j An example of a neutral peptide is (NANP) The primary sequence of (NANP) 3 is shown in Figure 1 and a Hopp and Woods profile is shown in Figure The amphipathic peptide for use in the hybrid peptide is the same as that described above for the first embodiment of the immunogenic composite.

SIn particular, the amphipathic peptides T *(HIVenv(112-124), H-E-D-I-I-S-L-W-N-Q-S-L-K) and T j (HIVenv(428-443), L-Q-I-I-N-M-W-Q-E-V-L-A-M-Y-A-NH i (Cease, et al., Proc. Natl. Acad. Sci. U.S.A.

84:4249-4253 (1987)) are useful in conjunction with hydrophilic or neutral peptides from HIV(e.g.

HIV(299-329)) for inducing an immune response to these non-immunigenic peptides.

The hybrid peptides can be made by any one nf the accepted state of the art peptide synthesizing reactions, either manually or in an automated peptide synthesizer. Also hybrid peptides can be prodiiued by standard state of the art genetic engineering procedures. In addition hybrid peptides can be produced by chemical or enzymatic buildup from peptide fragments or by cleavage of larger hybrid polyppptides or proteins.

Further, according to this embodiment nf the invention, any number and types of peptides ran be fused together to give an immunogenic composite to .i 1 16 which multiple antibodies are made. Of course, there is a practical limit on the size of the ultimate peptide in that there are technical limitations to the Slength of creating a synthetic peptide, as well as structural problems based on peptide folding should the peptide be too long or two short. These limitations can readily be determined by the skilled artisan.

The thus prepared hybrid peptide can be bonded to the lipid to form an immunogenic peptide-lipid complex "I by the same methods described above for the first embodiment of the immunogenic composite without regard to which of the peptides forming the hybrid peptide forms the covalent bond to the lipid.

i Examples of hybrid peptides include: HIV(469-511), comprised of the non-immunogenic hydrophilic peptide HIV(469-485) and the non-immunogenic amphipathic peptide HIV(487-511), and HIV(487-511), comprised of the non-immunogenic hydrophilic peptide HIV(500-511) and the nonimmuinogenic S amphipathic peptide HIV(487-499).

When HIV(469-511) is made part of an immiinngenic composite according to the present invention antibodies are produced against the non-immunogenic hydrnphilic peptide HIV(469-485) (as well as against the peptides HIV(487-511), (469-511), and (500-511)).

When HIV(487-511) is made part of an immunogenic composite according to the present invention.

antibodies are produced against the non-imminngenic hydrophilic peptide HIV(500-511) (as well as aqainst the peptides HIV(487-511) and (469-511)).

-26 The other of the two composites according to the present invention which are capable of selectively inducing or enhancing antibody production to a non-immunogenic hydrophilic and/or a non-immunogenic neutral peptide, as well as to a non-immunogenic amphipathic peptide, comprises a first peptide-lipid complex, wherein the peptide is at least partly comprised of an amphipathic peptide, and one or more additional peptide-lipid complexes associated with a mixture of one'or more lipids and one or more sterols.

*The first peptide-lipid complex comprises one of the above-described immunogenic complexes having an S amphipathic peptide associated therewith.

That is, the first peptide-lipid complex can be either the above-described peptide-lipid complex comprising a non-immunogenic amphipathic peptide covalently bound to a lipid or the above-described hybrid peptide-lipid complex comprising a hybrid peptide covalently bound to a lipid, wherein the hybrid peptide comprises a non-immunogenic amphipathic peptide covalently bound to one of: a non-immunogenic S* hydrophilic peptide; a non-immunogenic neutral peptide; a non-immunogenic amphipathic peptide: and a hybrid peptide comprising any combination of two or more of the above-defined peptides and The one or more additional peptide-lipid rnmplexes comprises one or more of the following complexes: a non-immunogenic hydrophilic peptide covalently bnind to I a lipid; a non-immunogenic neutral peptide covalently bound to a lipid; a non-immunngenic amphipathic peptide covalently bound to a lipid: and -27 1%^ p* _Y~I I I 1 a hybrid peptide covalently bound to a lipid, wherein the hybrid peptide comprises a non-immunogenic amphipathic, hydrophilic, or neutral peptide covalently bound to a peptide selected from: a non-immunogenic hydrophilic peptide; a non-immunogenic neutral peptide; a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two or more of the above-defined peptides and under the proviso that the amphipathic peptide is not albumin.

That is, the one or more additional peptide-lipid complexes can be any of those described previously, but in addition can include peptide-lipid complexes that do not have any amphipathic peptides.

According to this embodiment of the present invention, antibodies can be made to peptide-lipid complexes which are not, in and of themselves, capable of being immunogenic, any complex that does not include an amphipathic peptide.

Thus, for example, an immunogenic composite comprising a mixture of one or more lipids and one or more sterols having associated therewith one peptidelipid complex wherein the peptide is an amphipathic peptide such as T 2 or T 1 from HIV and another peptide-lipid complex wherein the peptide is a hydrophilic peptide such as HIV(299-329), will induce antibodies not only to the amphipathic "20 peptides T 2 or T 1 but also to the hydrophilic peptide HIV(299-329). If another peptide-lipid complex is included, antibodies to the peptide(s) in the additional complex will be induced.

This immunogenic composite, according to the third embodiment of the present invention, can be prepared in

S**

S -28oe

W'

the same manner as described above for the other immunogenic composites, except that two or more peptide-lipid complexes are associated with the mixture of one or more lipids and one or more sterols. This result can be achieved by, for example, reacting more than one peptide at the same time with the derivatized lipid, according to either of the two above-described methods.

The hydrophilic or neutral peptide for use in peptide-lipid complexes comprising a non-immunogenic hydrophilic peptide covalently bound to a lipid, a non-immunogenic neutral peptide covalently bound to a lipid and a hybrid peptide covalently bound to a lipid, is the same as that described above for the hybrid peptide, and the amphipathic peptide for use in any of the peptide-lipid complexes employing amphipathic peptides is the same as that described above for the first embodiment of the immunogenic composite.

The hydrophilic and neutral peptides are covalently bonded to the lipid in the same manner as described above for the amphipathic peptide-lipid complex.

Further, according to the present inventinn, any two or more of the above-described immllnngenic composites can be combined in admixture to qive an immunogenic composition. This approach can also be used to produce antibodies to more than one peptide.

The immunogenic composites according to the present invention are useful as immunogens. Prartical applications include non-clinical as well as clinical.

uses. Examples of non-clinical uses include -29

S

i

V

immunization for the purposes of raising monoclonal antibodies and producing antibodies to defined and specific epitopes for use in diagnostics.

Examples of clinical uses include the preparation of vaccines and induction of systemic or mucosal antibodies against sperm for use as a contraceptive.

Accordingly, the present invention also provides a pharmaceutical composition and a method for selectively inducing or enhancing antibody production.

The pharmaceutical composition comprises a mixture S* of one or more of the above-described immunogenic composites and a pharmaceutically acceptable carrier, diluent or excipient.

The method comprises administering a S* pharmaceutically effective amount of the pharmaceutical composition to a host in which an immune response is the normal means of defense to infection by foreign materials.

Examples of suitable pharmaceutically acceptable carriers, diluents and excipients include, for example, I* balanced salt solutions, any aqueous buffer, water and any of numerous inert carriers.

The dosage form can be oral, nasal, intramniirular.

intravenous, intraperitoneal, intranrular, subcutaneous, intravaginal, or on any mucosal surface.

Although the dosage varies with the nature nf the infectious agent against which immunity is sought and/or with the nature of the peptide against which antibody production is sought, one skilled in the art can readily determine suitable dosages.

A suitable dosage in mice varies from about 10 to about 100 pg per gm body weight.

EXAMPLES

The invention will now be described by reference to specific examples, but the invention should not be construed as being limited thereto.

I Unless otherwise specified, all percents, ratios, etc. are by weight.

EXAMPLE 1 see PRODUCTION OF IMMUNOGENIC COMPOSITE CAPABLE OF *1 .SELECTIVELY INDUCING ANTIBODY PRODUCTION TO i o NON-IMMUNOGENIC AMPHIPATHIC PEPTIDES o Initial Preparations Of Peptide-lipid Complex I. Joining of crosslinking reagent to phosphotidylethanolamine was accomplished acnording to I the method of Martin and Papahadjopoulos Biol.

Chem. 257:286-288 (1982)) as follows.

A. Reagents

S

1. Succinimidyl-4-(p-maleimidnphenyl) butyrate (SMPB) 2. Phosphatidylethanolamine (FE) 3. Anhydrous methanol 4. Triethylamine B. Procedure 1. Reagents were reacted for two hours Sat room temperature under N 2 2ml methannl. 5 p] triethylamine, 17 mg SMPB, added to 25 mg dry PE) -31 0 2. Methanol was removed from the reaction products with a stream of N 2 3. 2 ml CHCL 3 was added and the mixture was extracted 2 times with 2 ml of 1% NaCl in H20 by centrifuging at 1500 RPM, 5 min. All procedures in this step were performed under N 2 4. Chromatography on a silica gel column was then performed as follows: a. 'The column was washed with ml CHC1 3 3' b. The sample in 5 ml CHCI. was loaded on the column, followed by 10 ml each of CHC1 3 S MeOH 40:1, 30:1, 25:1, 20:1, 15:1, and then 40 ml 10:1 while collecting 5 ml fractions.

5. Thin layer .chromatography of the silica gel column fractions (solvent-CHCl :MeOH:H 2 0, S65:25:4) was then conducted. The coupled cross linker and phospholipid (MPB-PE) that eluted in the early 10:1, CHCI :MeOH fractions showed a single spot 3 comigrating with a PE standard. The derivati7.ed PE spot was ninhydrin negative, iodine positive. The PE standard spot was ninhydrin positive. Silira gel i column fractions containing MPB-PE were pniled and stored under N 2 at -20 0

C.

6. Phospholipid was quantitat- hy the method of Bartlett Biol. Chem. 234:466 (1q5 9 The ave. yield was II. Reduction of peptide according tn Roche procedure A. Reaction -32 .b *W* -r <r 1. The amphipathic peptide HIV(487-511), HIV(469-511), or HIV(578-608), prepared by solid-phase procedures (Yarney and Marrifield. In "The Peptide: Analysis, Synthesis, Biology." Academic Press, N.Y.

Vol. 2 1-284.) was dissolved in 1M acetic acid (HAc) 2 mg peptide in 1 ml HAc).

2. Dithiothreitol (DTT) was added to 100 mM conc.

3. The sample was degassed, nitrogenized, and then incubated in a screw-cap tube at 370 for 3 hours.

B. Affinity chromatography on Bondelute column (3cc column obtained from Analytichem International C18, part 607203).

1. The column was washed with 2 vols 100% I MeOH, then 10 vols IMHAc.

2. The peptide was applied to the Bondelute column.

I 3. The column was washed with 5 volumes 1M HAc, then 5 vols 0.02% trifluoracetic acid (TFA) 4. The sample was eluted with 5 volnuims acetonitrile, 0.02% TFA.

i 5. The eluted sample was lyophilized overnight and stored at -20 0 C. Essentially r-'mplete recovery was obtained.

III. Preparation of immunogenic rnmprsites comprising peptide-lipid complex associated with a mixture of lipids and a sterol (cholesterol) A. Lipids MPB-PE, sphingomyelin (SP).

I phosphatidylcholine phosphatidylserine (PS) Sterol cholesterol (Ch) -33

I

B. Procedure 1. The lipids and cholesterol (Ch) were dissolved in ether (10 mg/ml) at a molar ratio of MPB-PE:SP:PC:PS:Ch of 2:1:1:1:5.

j 2. The sample was dried under a nitrogen stream.

3. The dried sample was resuspended in buffer (20 mM citric acid, 35 mM disodium phosphate, 108 mM NaCI, 1 mM EDTA, pH 4.5) at 4 mg lipid and cholesterol/ml.

4. The sample was sonicated to form a particulate suspension.

I 5. Octyl-B-D-glucoside (10 mg/mg lipid S. and sterol) was added to the sonicated sample.

6. The sample was again sonicated to I dissolve all lipid.

7. The sample was stored under N2 in the dark.

IV. Coupling of peptide to lipid mixture containing derivatized phosphatidytethannlamine

.(MPB-PE)

i A. Procedure 1. Premade reduced peptide from step II above was added to the lipid/sterol mixture from step III above containing MPB-PE (MPB-PE molar concentration was 2x molar concentration of peptide) at pH 2. The pH was adjusted to 6.5 with IN NaOH.

3. The MPB-PE and peptide in the mixture were allowed to react under N 2 at room temperature overnight.

-34 several changes 7.2.

the protein-lipi lipids and cholE bag and stored r PRODUCTION C

SELECTIVEL

NON-IMM

V"

The mixture was dialyzed against of phosphate buffered saline at 4 0 C, pH The immunogenic composite comprising id complex associated with a mixture of esterol was recovered from the dialysis efrigerated (4 0

C).

EXAMPLE 2 )F IMMUNOGENIC COMPOSITES CAPABLE OF ,Y INDUCING ANTIBODY PRODUCTION TO UNOGENIC HYDROPHILIC'PEPTIDES Immunogenic composites capable of selectively inducing non-immunogenic neutral or hydrophilic I peptides were made in two ways: constructing the neutral or hydrophilic peptide contiguous to an amphipathic peptide to form a hybrid peptide by standard procedures as described in, for example Varney, G. and Marrifield, R.B. in The Peptides: Analysis, Synthesis, Biology, Gross J.

Meinhofer, Eds.) Academic Press, N.Y. 2:1-284. reducing the hybrid peptide, and then covalently bonding the reduced hybrid peptide to a derivatized lipid present in solution with other lipids and cholesternl as described above in Example 1 and dialyzing as d =rribed in Example 1 to form an association of the hybrid peptide-lipid complex with a mixture of lipids and cholesterol; or covalently bonding a mixture of a reduced non-immunogenic neutral or hydrophilic p-ptide j Lt and a reduced amphipathic peptide to a derivatized lipid present in solution with other lipids and cholesterol as described in Example 1, and dialyzing as described in Example 1 to form an association of both peptide-lipid complexes with a mixture of lipids and a sterol.

Using method 1, immunogenic composites were prepared using the HIV peptides (487-511). (469-511) and (578-608).

EXAMPLE 3 ANTIBODY PRODUCTION BY IMMUNOGENIC COMPOSITES OF EXAMPLE 1 Six to eight week old female inbred mice were immunized intraperitoneally with the immunogenic composites prepared in Example 1 (and composites which were prepared as in Example 1 but where the peptide was (NANP) IL-2(1-12)-trans (the primary aminn acid sequence is shown in Figure HIV env(469-485), HIV env(500-511) and HIV env(578-608) all of which are either hydrophilic or neutral). Each was used in an amount equivalent to 30 pg of peptide on days 0 and 14.

On day 28 mice were immunized with the compnsitp- in an amount equivalent to 7.5 pg of peptide. Mi'o were bled from the retroorbital socket on days 0. 34. 28 and 42. Following collection, the blood was all nwed to clot and the serum was separated by centrifuiqtion.

Serum was tested for antibody by the enzyme linked immunosorbent assay (ELISA) described below.

ELISA Direct Binding Assay -36 It

V

r A. Plate Coating 1. Wells were coated with antigen (whichever peptide was used to immunize or to test against) diluted to about 0.5 to 1 pg/ml in bicarbonate-carbonate saline, pH 9.6 2. Wells were coated with BSA or gelatin diluted to lOx the concentration of antigen for control.

100 p1 of BSA in bicarbonatecarbonate buffer were placed in each well.

Plates were dried overnight at Seg 37 0

C.

S(c) Wells were washed 5 times with dH20 or PBS/tween (3x).

Blocking Reaction 200 pl of 1% gelatin in Borate Buffered saline, pH 8.2 or 1% BSA in PBS, pH 7.0 (no Stween) were placed in each well.

Plates were covered and incubated 1 hour at 37 0

C.

After incubation, wells were washed 5 times with Ix PBS/tween.

C. Binding Reaction Serum containing antibody was dilnted (a series of 2-fold dilutions starting at 1:100) with 1% BSA in PBS (0.5 M NaCI) and tween, pH 100 pl of sample was arded per well and the plates were covered.

Samples were incubated at FT on a shaker for 2 hours.

-37

I

3~l~ai Plates were washed 5 times with PBS/tween.

D. Detection Reaction for Mouse Antibodies 1. Affinity purified goat antimouse IgG and IgM-HrP (BOEH. MANN. Cat. 605-26) was diluted 1:1000 in 1% BSA in PBS/tween, pH6.

100 pl of the solution was added to each well, and the plates were covered and protected from light.

The solutions were incubated at RT on a shaker for 2 hours.

The plates were washed 5 times with PBS/tween.

SE. Substrate Reaction 1. O-phenylenediamine dihydrochloride S* (SIGMA P-1526) was mixed in 0.1 M citrate buffer. pH to give a final concentration of 0.4 mg/ml. 4 pl of 30% H202 was added for every 10 ml of buffer.

100 pl of the solution was added to each well, and the plates were proterted from o, light.

h The solution was allowed to react for 15 min. at RT on a shaker.

The extent of antibody binding was determined by measuring the optical density at 488 nm.

Solutions for ELISA Bicarbonate-carbonate Buffer M NaHCO 3 M Na2CO 3 -38

LI

mix in approximately equal volumes until PH 9.6.

Bicarbonate-carbonate Saline mis lOx B-C buffer mis 5M NaCi 1.8 mls 1M NaN 3 bring to 250 mls with distilled H-i 2 0 and filter (0.20 p pore size).

1% BSA in PBS, PH mis 0.5 M NaPO buffer, pH 15 mis SM NaCi 0.5 mlAIM MgCI 2 1.0 ml 0.5M CaCl 2 5.0 gins Bovine serum albumin, Fraction V. Reagent Grade tw me Bring to 500 mis with dH 0 and filter (0.45 p pore size).

a 4 Store at 4'C.

*0.5 M NaPO Buffer 4 1. 2 aHP0 4 '7H20 (dibasic) -67 gin/5QO ml NaH 2 PO 4 .H 2 0 (monobasic) 34.5 gm/500 ml Mix and together until desired pH is reached. For pH 6.5 add approximately equal vonlumes of 1" and For pH 7.0 add 500 ml to -200 ml 1% BSA in PBS/tween. pH mls 0.5 M NaPO 4 buffer, pH mls 5M NaCI ml 1M MgCI 2 1.25 ml 20% ml 0.5M CaCl 2 -39gmn Bovine serum albumin, Fraction V, Reagent Grade Bring to 500 mis with dH 20 and filter (0.45 p pore size).

Store at 4 0

C.

BSA in PBS (0.5 M NaCI) and tween, _pj mis 0.5M NaP'0 buffer, pH ntis SM NaCi ml iM MgCI 2 1.25 ml 20% 1.0 ml 0.5M CaCI 2 9G* 2 5.0 gmn Bovine serum albumin, Fraction V, Reagent grade Bring to 500 mis with dH 20 and filter (0.45 p pore size).

Store at 4 0

C.

0.1M Citrate Buffer, PH 7.35 gmn trisodium citrate 0 *2 H0) 5.25 gin citric acid monohydrate Bring to 500 ml, pH HS and 50mM NaO (sdim-et 2-4 2 q 2 5 soim et bisulfite) 69.4 ml conc H 2so4 4.75 gm sodium meta bisulfite Mix each separately in dH 2 0. Add togeth-r Bring to 500 mis with dH 2 O0 and filter (0.41;1 pore size).

Store at 4 0

C.

1% gelatin in BBS, pH 8.2 gins gelatin 6.18 gin H 3 BO0 3 Bringm to I iter with dH 0 and filter (0.45 p pore 9.54 g Na 2 2 size).

Store at RT.

2% BSA in PBS (IM NaCI) and 0.15Y. Tween-20 ,p 6.

gins Bovine serum albumin, Fraction V, Reagent grade 100 mis SM NaCi mis 0.5M NaPO 4 buffer, pH iM MgCl 2 *1.0 ml 0.5M CaCl 2 3.75 ml 20% Bring to 500 mis with dH 0 and filter (0.45 p pore 2 size).

Store at 4 0

C.

The results for sera obtained on day 42 after initial immunization are shown in Table 1 belnw.

-41 000 9 Table 1 Antibody Titers Of Mice Immunized With Various Peptide-Lip~id Coniucqates Log 10 Antibody Titer to Immunizing Peptide Peptide 0*

S

S. S S *5

S.

9* 9 0O@SSS S. S S 56

S.

S.

S*

*SS*

S 9* 5

S

S..

Hydrophilic or Neutral (NANP 3 IL-2 (1-12)-trans HIV env. (469-485) HIV env. (500-511) HIV env. (647-659) 0.0(6)* 1.2 1.3 (6) 0.0(5) 0.0(5) 0.0(5) Amphipathic HIV env.

HIV env.

HIV env.

(469-511) (487-511) (578-608) 4. 4 4.3 8(4) 4. 4 the figure in parenthesis indicate-s Hp number of mice per gro~up.

The results clearly show that complexing an amphipathic peptide with phospholipid and assnriat-ing it with additional phospholipid and cholesterol induces a significant immune response when injected into mice.

This is in contrast to the results with neutral anid hydrophilic peptides which do not induce an immiin, -42 response in mice even when covalently linked to phosopholipid and complexed with additional phospholipid and cholesterol.

That the amphipathic peptide is not by itself immunogenic is seen from the data set forth in Table 4 of Example EXAMPLE 4 ANTIBODY PRODUCTION BY IMMUNOGENIC COMPOSITES OF EXAMPLE 2 Mice were immunized with the immunogenic composites prepared in Example 2 and with composites prepared as in Example 1 but using HIV peptides (469-485) and (500-511). Antibody titers were determined as in Example 3.

The results are shown in Table 2 below.

6* q *S S -43

I

611.,- i .i 1.

i Table 2 Immune Response To Nonimmunogenic Hydrophilic Peptides Of HIV Envelope Protein When Synthesized Contiguous With A Hydrophobic Sequence From The Same Protein Immunizing Peptide 469-485(5)* 487-511(5) 469-511(5) 500-511(5) 469-485 0.0 0.0 2.810. 7 0.0 Logl0 Antibody Titer to 487-511 469-511 500-511 0.0 0.0 0.0 5.3±0.2 3.2±0.8 3.2+0.5 3.2±0.8 4.4±0.5 3.1+0.8 0.0 0.0 0.0 *0 S

S

S.

9* 9 S. S

S

S

*5

S

*5 0 5

S

the figure in parenthesis indicates the number of mice per group.

The results show that when non-immunogenic peptides (469-486, 500-511) were synthesized contiguous to amphipathic peptides (in 469-511 and 487-511) the mice were able to produce antibodies to the neutral and hydrophilic regions. Thus, these regions became immunogenic when incoporated into larger amphipathic peptides, complexed to phospholipid, and asnr'iated with additional phospholipid and cholesterol.

As a method of adding amphipathic strnr-tures, purified viral glycoproteins Gould-fogerite and J.

Mannino, Anal. Biochem. 148:15-26 (1985)) wprp added.

by mixing with dissolved peptide-lipid, phosphnlipids and simple lipids, to some of the nonimmlnngenic peptide-phospholipid complexes at 1/200 f their naturally occurring concentrations and the rnmposite -44 27 9 was dialyzed overnight at room temperature against 3 changes of phosphate-buffered saline pH 7.2.

The results are shown in Table 3 below.

Table 3 Effect Of Addition Of Highly Immunogenic Protein~s To Liposomes Containing Nonimmunogjenic Peptide Phospholipid Conjugate s 00 0 0 000 0 0@ *0 0000 S S *0 0 000000 0 00 0 00 @0 *5 00 0 0* *6 0 6 0000 0e 0* S @0 @00 Peptide (NANP 3 (NANP 3 (NANP 3 IL-2 (1-12)-trans IL-2 (1-12)-trans IL-2( 1-12)-trans Viral Glycoproteins Influenza Sendai Influenza Senda i Influenza Senda i Log 10 Antibody Titer to Immunizing Peptide 0.0* 0.0(6) 0.0(6) 3 2(6) 3 5 1.2±1.3(6) 3 .5±0.3(F6)) 4.1±0.4(6) Antibody titers to Sendai and influenza glyrnpr'nteins were greater than log 10 4.5 in all mice tested.

No antibodies were detected to either (MANP) 3 or IL-2 (1-12)-trans **The figure in parenthesis indicates thait nuimber of mice per group. Ir As shown above in Table 3, the adciitinn of amphipathic molecules in the form of viral glycoproteins results in the production of antibodies i to two previously nonimmunogenic peptides. In each case, the increase in antibody titer is greater than 2 logs.

Therefore, when neutral or hydrophilic peptides are complexed to phospholipid and added to preparations containing amphipathic structures and additional phospholipid and cholesterol, they become immunogenic.

EXAMPLE IMPORTANCE OF COUPLING NON-IMMUNOGENIC PEPTIDES TO LIPIDS IN ORDER TO PRODUCE IMMUNOGENIC RESPONSE *S e Table 4 below shows the importance of coupling the amphipathic peptide to lipids in order to produce an immunogenic structure.

-46* 0* 0 -46 11 Table 4 Loglo Antibody Titer to Peptide Immunizing Peptide SHIV env. (487-511) i mixed lipid conjugate 5.3±0.2(5) i HIV env. (487-511) mixed lipid conjugate 5.4±0.3(5) SHIV env. (487-511) 1I mixed lipid free 0.0(5) i HIV env. (487-511) in sterile PBS 0.0(5) i Antibody titers of mice immunized with HIV env.

(487-511) conjugated to phospholipid by th- following i methods: MPB-PE was mixed with the other phospholipids and cholesterol in small unilamelar vesicles S. and reacted with peptide overnight at room 1 temperature. The reaction mixture was ji dissolved with octyl-8-D-glucoside (10 mg/mg lipid) and dialyzed against CMF-PBS to form a Speptide phospholipid complex.

or i MPB-PE and mixed phospholipids and cholesterol were dissolved in Octyl-B-Dglucoside (10 mg octyl-B-D-glu-ide/mg lipid), reacted with peptide overnight at room temperature and subsequently dialyzed against CMF-PBS to form a peptide phospholipid complex.

or -47

K

A

uncomplexed and mixed with preformed lipid-cholesterol structures or uncomplexed and injected in sterile saline.

The results in Table 4 show that when the peptide is complexed to phospholipid as a lipid and associated with additional phospholipid as a lipid and cholesterol as a sterol, an immunogenic composite is produced.

This composite is significantly immunogenic regardless of whether the initial coupling is to MPB-PE already associated in a structure with other lipid and cholesterol or free in solution by virtue of being dissolved in octyl-B-D-glucoside. In contrast, if-the peptide is mixed with preformed lipid-cholesterol S structures, but not complexed with phospholipid no S immune response is observed. Furthermore, no immune response is observed if solubilized 487-511 is injected into an animal in sterile PBS. Therefore, it is of utmost importance for the amphipathic peptide to be covalently linked to the lipid for it to be immunogenic.

EXAMPLE 6 CONSTRUCTION OF A SYNTHETIC VACCINE AGAINST MALARIA The peptide (NANP) is a repeated peptidp of greqt immunological significance in protection from infprtion by malaria sporozoites. However, (NANP) is a neutral peptide and is not in and of itself immunogenic in most animals.

Recently, an immunologically important amphipathic peptide, also from the malaria sporozoite, ha.~ been -48 identified Good, et al., Science 235:1059-1062 (1987). This amphipathic peptide has the following sequence: P-S-D-K-H-I-E-Q-Y-L-K-K-I-K-N-S-I-S.

The vaccine would be produced as follows.

The fused peptide, P-S-D-K-H-I-E-Q-Y-L-K-K-I- K-N-S-I-S-(NANP) n=3, is constructed in a synthetic protein synthesizer.

The fused peptide is then reduced with dithiothreitol and solubilized in a non-ionic detergent with a high critical micelle concentration, such as octyl-B-D-glycoside, along with derivatized phosphatidylethanolamine a mixture of lipids comprising, for example sphingomyelin, phosphatidylserine, and phosphatidylcholine. and cholesterol as a sterol.

The fused peptide is cross-linked to the derivatized PE by one of the know:- methods, such as by that of Martin and Papahadjopoulis (1982). J. Biol.

Chem. 257:286-288.

The solution is dialyzed against phosphate-buffered saline, pH 7.2 to give a partirulate suspension of an immunogenic composite and the suspension is adjusted to the appropriate conrpn ation in phosphate buffered saline.

EXAMPLE 7 CONSTRUCTION OF A SYNTHETIC VACCINE AGAINST HIV III The following non-immunogenic hydrophilir peptides of immunologic significance are known for HTV III: (299-329) H-R-P-N-N-N-T-R-K-I-R-I-R-E-P-E-R-A-E W I-E- -49

I'

N-M-R-Q-C-NH and (735-752) D-R-P-E-G-I-E-E-E-G-G-E-Rf D-R-S.

The following immunologically important amphipathic peptides also from HIV III have recently been described by K.B. Cease (Proc. Nat. Acad. Sci. USA 84:4249-4253 (June 1987)): T 2 (HIVenv(112-124).

H-E-D-I-I-S-L-W-H-Q-S-L-K) T 1 (HIVenv(428-443),

L-Q-I-I-N-M-W-Q-E-V-L-A-M-X-A-NH

2 and by Putney et al.

(Science 234:1392-1395 (Dec. 1986)): (HIV(340-368)

H-N-N-T-L-K-Q-I-D-S-K-L-R-E-Q-F-G-N-N-L-Q-S-S-G-C-NH).

One or more vaccines would be produced according S to the procedures of the present invention by forming immunogenic composites with protein-lipid complexes wherein the protein is as follows: 340-368 alone; 340-368 fused to 487-511; 299-324 fused to 340-368; 299-324 fused to 487-511; 299-324 fused to 578-608: and 340-368 fused to 578-608.

.i *More preferably, immunogenic composites are formed which include more than one of the above-described protein-lipid complexes, e.g. 340-368 fused to 4P7-511 and 299-324 fused to 378-608 are included in the same composite.

I.

While the invention has been described in detail and with reference to specific embodiments ther=-e, it will be apparent to one skilled in the art that v'rious changes and modifications can be made therein without departing from the spirit and scope thereof.

lt

Claims (5)

1. An immunogenic composite capable of selectively inducing or enhancing antibody production to a non-immunogenic peptide comprising a peptide-lipid complex which comprises a non-immunogenic amphipathic peptide covalently bound to a lipid, under the proviso that the amphipathic peptide is not albumin.

2. The immunogenic composite of claim 1, wherein said peptide-lipid complex is further associated with a mixture of one or more lipids and one or more sterols.

3. An immunogenic composite capable of selectively inducing or enhancing antibody production to one or more non-immunogenic peptides comprising a peptide-lipid complex which comprises a hybrid peptide covalently bound to a lipid and said hybrid peptide comprises a non-immunogenic amphipathic peptide covalently bound to one peptide selected from the group consisting of: a non-immunogenic hydrophilic peptide; a non-immunogenic neutral peptide; a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two or more of the above-defined peptides and under the proviso that the amphipathic peptide is not albumin.

4. The immunogenic composite of claim 3, wherein said peptide-lipid complex is further associated with a mixture of one or more lipids and one or more sterols. An immunogenic composite capable of selectively inducing or enhancing antibody production to one or more non-immunogenic peptides comprising a T- 51 S S 5* S I 1' S SS

455. 0 0 0 S* S* *c S S. first peptide-lipid complex and one or more additional peptide-lipid complexes associated with a mixtuire of one or more lipids and one or more sterols, wherein said first peptide-lipid complex comprises one member selected from the group consisting of: a non-immunogenic amphipathic peptide covalently bound to a lipid; and a hybrid peptide covalently bound to a lipid, wherein said hybrid peptide comprises a non-immunogenic amphipathic peptide covalently bound to peptide selected from the group consisting of: a non-immunogenic hydrophilic peptide; a non-immunogenic neutral peptide: a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two or more of the above-defined peptides and and said one or more additional peptide-lipid complexes comprises one or more members selected from the group consisting of: a non-immunogenic hydrophilic peptide covalently bound to a lipid; a non-immunogenic neutral peptide cnrvalntly bound to a lipid; a non-immunogenic amphipathic peptidp covalently bound to a lipid; and a hybrid peptide covalently bound to a lipid. wherein said hybrid peptide comprises a non-imminngeni.r amphipathic, hydrophilic or neutral peptide rnv lently bound to peptide selected from the group consisting of: -52 a non-immunogenic hydrophilic peptide; a non-immunogenic neutral peptide; a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two or more of the above-defined peptides and under the proviso that the amphipathic peptide is not albumin. 6. The immunogenic composite of claim 3 or claim 4, wherein said hybrid peptide comprises said non-immunogenic hydrophilic peptide covalently bound to said non-immunogenic amphipathic peptide. 7. The immunogenic composite of claim 3 or claim 4, wherein said hybrid peptide comprises said non-immunogenic neutral peptide covalently bound to said non-immunogenic amphipathic peptide. 8. The immunogenic composite of claim 5, wherein said one or more additional peptide-lipid complexes comprises said non-immunogenic hydrophilic peptide crosslinked to a lipid. ea.* I 9. The immunogenic composite of claim 5, wherein said one or more additional peptide-lipid complexes comprises said non-immunogenic neutral peptide crosslinked to a lipid. The immunogenic composite of any one of claims 1 to 4, wherein said lipid in said peptide-lipid complex is phosphatidylethanolamine. 11. The immunogenic composite of claim 5, wherein said lipid in one or more of said peptide-lipid complexes is phosphatidylethanolamine. 53 I 12. The immunogenic composite of claim 2, 4 or 5, wherein said sterol is cholesterol. 13. An immunogenic composition comprising a mixture of two or more immunogenic composites as defined in any one of claims 1 to 14. An immunogenic composite capable of selectively inducing or enhancing antibody production to a non-immunogenic peptide comprising a peptide-lipid complex which comprises peptides derived from a human immuno deficiency virus, HIV, envelope protein covalently bound to a lipid. An immunogenic composite of claim 14, wherein said human immuno deficiency virus, HIV, envelope protein is selected from the group including HIV(487-511), HIV(469-511), HIV(578-608), HIV(469-485), HIV(500-511) or HIV(647- 659). 16. An immunogenic composite of any one of claims 1 to 15, substantially as hereindescribed with reference to any one of the Examples and accompanying figures. A. 17. A pharmaceutical composition for selectively inducing or enhancing antibody production comprising a mixture of: one or more immunogenic composites selected from the group 1 consisting of: 54- 54 !f r F peptide-lipid complex which comprises a non-immunogenic amphipathic peptide covalently bound to a lipid; (ii) QO peptide-lipid complex associated vwth a mixture of one or more lipids and a one or more sterols, wherein said peptide-lipid complex comprises said non-immunogenic amphipathic peptide covalently bound to a lipid; (iii) a peptide-lipid complex which comprises a hybrid peptide covalently bound to a lipid and said hybrid peptide comprises a non-immunogenic amphipathic peptide .covalently bound to one peptide selected from the group consisting of: a non-immunogenic hydrophilic peptide; a non-immunogenic neutral S. peptide; a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two or more of the above-defined peptides (b) and 000 (iv) o- peptide-lipid complex associat- with a mixture of one or more lipids and one nr more sterols, wherein said peptide-lipid complex comprises a hybrid peptide covalently bound to a lipid and said hybrid peptide comprises a non-immunogenic amphipathic peptide covalently bound to one peptide seleict-d from the group consisting of: a non-immunogenic hydrophilir p ,l~q 7"E SI bar peptide; a non-immunogenic neutral peptide; a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two or more of the above-defined peptides and and a first peptide-lipid complex and one or o. more additional peptide-lipid complexes associated with a mixture of one or more lipids and one or more sterols, wherein said first peptide-lipid complex comprises one member selected from the group consisting of: a non-immunogenic amphipathic peptide covalently bound to a lipid; and a hybrid peptide covalently bound to a lipid, wherein said hybrid peptide comprises a non-immunogenic amphipathic peptide covalently bonnd to peptide selected from the group consisting of: a non-immunogenic hydrophilic S: peptide; a non-immunogenic neit-ral peptide; a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two nr more of the above-defined ppt-ides and -56 -I and said one or more additional peptide-lipid complexes comprises one or more members selected from the group consisting of: a non-immunogenic hydrophilic peptide covalently bound to a lipid; a non-immunogenic neutral peptide covalently bound to a lipid; a non-immunogenic amphipathic peptide covalently bound to a lipid; and a hybrid peptide covalently bound to a lipid, wherein said hybrid peptide comprises a non-immunogenic amphipathic, hydrophilic or neutral peptide covalently bound to peptide selected from the group consisting of: a non-immunogenic hydrophilic peptide; a non-immunogenic neutral peptide; a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two or more of the above-defined peptides and and a pharmaceutically acceptable carrier, diluent or excipient, under the proviso that the amphipathic peptide is not albumin. 18. The pharmaceutical composition of claim 17, wherein said lipid in said peptide-lipid complexes is phosphatidylethanolamine. 19. The pharmaceutical composition of claim 17, wherein said sterol in said Simmunogenic composites (iv) and is cholesterol. A pharmaceutical composition of claims 17 to 19, substantially as hereindescribed with reference to any one of Examples 3 to 7. W- 57- AR 21. A method for selectively inducing or enhancing antibody production comprising administering to a host, in which an immune response is the normal means of defense to infection by foreign materials. one or more immunogenic composites selected from the group Sconsisting of: a peptide-lipid complex which comprises a non-immunogenic Samphipathic peptide covalently bound to a lipid; (ii) a peptide-lipid complex associated with a mixture of one or more lipids and a one or more sterols, wherein said peptide-lipid complex comprises said non-immunogenic amphipathic peptide covalently bound to a lipid; (iii) a peptide-lipid complex which comprises a hybrid peptide covalently bound to a lipid and said hybrid peptide comprises a non-immunogenic amphipathic peptide covalently bound to one peptide selected from the group consisting of: a non-immunogenic hydrophilic peptide; a non-immunogenic neutral peptide; a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two or more of the above-defined peptides and P t| ©i '58- 1i iO r S(c); (iv) Q_ peptide-lipid complex associated with a mixture of one or more lipids and one or more sterols, wherein said peptide-lipid complex comprises a hybrid peptide covalently bound to a lipid and said hybrid peptide comprises a non-immunogenic amphipathic peptide covalently bound to one peptide selected from the group consisting of: a non-immunogenic hydrophilic peptide; a non-immunogenic neutral 00 peptide; a non-immunogenic amphipathir peptide; and a hybrid peptide comprising any combination of two or more of the above-defined peptides and and a first peptide-lipid complex and nne or more additional peptide-lipid complexes associated with a mixture of one or more lipids and one or more sterols, wherein said first peptide-lipid complex comprises one member selected from the group rcnsisting of: a non-immunogenic amphipathir peptide covalently bound to a lipid; and a hybrid peptide covalently bound to a lipid, wherein said hybrid peptide comp ises a non-immunogenic amphipathic peptide covalently bhlnnd to peptide selected from the group consisting of: a non-immunogenic hydrophilic -59 1I 4 r^ 0S 6e S S S S *5 S S S. S. S *s peptide; a non-immunogenic neutral peptide; a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of two or more of the above-defined peptides and and said one or more additional peptide-lipid complexes comprises one or more members selected from the group consisting of: a non-immunogenic hydrophilic peptide covalently bound to a lipid; a non-immunogenic neutral peptide covalently bound to a lipid; a non-immunogenic amphipathic peptide c.ovalently bound to a lipid; and a hybrid peptide covalently bound to a lipid, wherein said hybrid peptide comprises a non-immunogenic amphipathic, hydrophilic or neintral peptide covalently bound to peptide selected from the group consisting of: a non-immunogenic hydrnphilic peptide; a non-immunogenic ne'ltr:l peptide; a non-immunogenic amphipathic peptide; and a hybrid peptide comprising any combination of twn nr more t 1 L, Sof the above-define peptides and and a pharmaceutically acceptable carrier diluent or excipient. 22. The method of claim 21, wherein said lipid in said peptide-lipid complexes is phosphatidylethanolamine. 23. The method of claim 21, wherein said sterol in said immunogenic composites (iv) and is cholesterol. 24. A method for selectively inducing or enhancing antibody production of claim 21 which method is substantially as hereindescribed with reference to any one of Examples 3 to 7. DATED this 3 day of June 1992. ALBANY MEDICAL COLLEGE By their Patent Attorneys: CALLINAN LAWRIE *C 61 tE Na-e Name Sequence (NAN P) 3 h -I L-2 -Y trans HIV (469-485) HIV (500-511) HIV (647-659) HIV (469-511) HIV (487-511) HIV (578-608) *0 9. Ac- C- N A- N P- N A- N P- N -A P -OH H-A-P -T-S-S-S-T-K-K-T-Q-L-Y-Q-V-A-V-A-G L-V- F-L- L-I L-L-L-S -G-L-T H-R-P-G- G-G-D-M-R-D-N-W-R-SELY-K-CNH 2 H-K-A-K-R-R-V-U-Q-R-E-K-R-C-NH 2 H -E -E S-9-N E K-N E -Q -E -C -NH 2 H-R-P-G-G-G-D-M-R-D-N E-L- Y- K-Y-K-Y V K- I- E-P L G-V -A -P T- K -A -K -R -R V R- E-K-R-C-NH 2 H K-1-E- P-L- G-V-A- P-T-K-A- K- R-R -V-V- 9-R-E-K-R-C-NH 2 H R-I- L-A-V- E- R-Y K-D Q-Q-L-L- G-I G C -S C K L-1I- I C T- T -A -V N H2 S I C14 >4 I 0 a. 0* 9s 00 0 S a *5 0* *0 0 0*tS*S 0 4( I H 0 *t- 4 *t-i H (I~ *t-q *t- 4 0 00 0 00 000 0 0 00 0 0 000*00 .0 0 0 0000 00.000 00 0 0 0 0000 I. a a. a a eS a 9' 'K I~4%'K 'K'9' .9' 99 -k -k 'K 9' *9c r '4c'9''K .9' 9' 'K 'K 'K ~9' 'K '9' 'K 'K '9' '9' 'K'w'K'K 'K.9' 'K '9' 'K 'K'9' IK" 4c g a a a. a a a. *aa.a. a *9 *K *K IK i MI. *K :zK'9 .9' a. a I M P N A N P N A N P N A N P N A N P N A N P N A N P (NANP) N -9 0 0 9* w9 9 *9099* .9 S a 0* a. *9* 9 9 0 a. S S a. 4*S*S* a